NMR spectroscopy has been found to be an important procedure in chemical analysis for many years. Since the end of the 1970s, it has been used to study living cells and tissues with increasing importance. It enables, inter alia, the measurement of metabolite concentrations, pH values and the kinematics of enzymatic reactions in intact cells and allows a researcher to follow metabolic processes.
A serious problem with the in vivo use of NMR spectroscopy for cell suspensions is that the method is relatively insensitive. For example, the measuring time required for a given signal/noise ratio (S/N) increases by a square law with decreasing cell density (number of cells per volume), i.e. the measuring time is in an inverse square relationship with the cell density. That means that a ten-fold reduction in the cell density requires a 100-fold greater measurement time. This, in turn, creates problems with respect to maintenance of the viable cell materials in an undamaged state in the measuring tubes over the lengthy measuring periods.
Upon research with cell suspensions, therefore, it has been desirable to concentrate them to the highest possible extent (for example, to 50% cell internal volume in terms of the total sample volume).
In K. D. Barrows et al (J. Biol. Chem. 259 (1984)-5711, 5716), for example, experiments are described in which the cell suspensions are concentrated to 34% internal cell volume. During the measurements, the organisms are retained in standard NMR tubules (10 mm diameter, 20 cm length) without concern for damage. In this manner, with a one-minute exposure time, spectra can be obtained at the important intracellular resonances with a signal/noise ratio (S/N) of about 5:1.
In order to maintain cells in such highly concentrated suspension for long periods of time in a stable state, attempts have been made to supply the cells in situ in the NMR measuring chamber. For this purpose, cylindrical so-called hollow-fiber reactors with a diameter of about 20 mm can be used. Here nutrients and waste products diffuse through the membrane walls of numerous hollow fibers traversed by the suspension (see R. Gonzalez-Mendez, et al, Biochim Biophys. Acta 720 (1982) 274-280). In such reactors, the cell can be retained for periods in excess of 40 days in a viable state. This state is not, however, constant either over the total time period nor over the entire reactor volume.
In H. Santos, et al (J. Magnetic Resonance 68 (1986) 345-349), a method of improving the sensitivity is described wherein the signal obtained by an effective reduced saturation of the NMR signal is obtained. In the experimental apparatus, a dense cellular suspension is continuously circulated with the aid of a air-lift system through the NMR measuring chamber which can be a standard NMR tubule with a diameter of 10 mm and a length of about 20 cm, containing a coaxial inner tubule. By comparison with standard conditions, the filling level of the 10 mm tubule is increased by a factor of 2 to 3. The portion of the NMR tube above the NMR measuring chamber is here used as a premagnetization chamber. The apparatus here described is used for in vivo .sup.13 C NMR with continuous flowthrough operation with a residence time in the measuring chamber greater than 4 seconds (maximum flow rate 1 ml/min:maximum chamber volume about 2 ml).
The use of a premagnetization chamber or space is also described in J. J. Grimaldi et al (Rev. Sci. Instr. 46 (1975), 1201-1205, for investigations into the kinematics of chemical reactions. Here the premagnetization volume is provided with the same diameter and approximately the same volume as the measuring chamber. This system, however, is used only for stop-flow operation, for, example .sup.1 H NMR.
E. Bayer et al (J. Chromat. 312 (1984) 91-97) describe the use of a premagnetization space or compartment which, however, is not described in detail. In the U-tube arrangement of this document, the measuring chamber has a greater cross section than the feed line. It is designed for .sup.13 C NMR in continuous flowthrough operation with a residence time in the NMR measuring chamber greater than 3 seconds.